Electrochemical detection of extracellular hydrogen peroxide in Arabidopsis thaliana: a real-time marker of oxidative stress

Abstract

An electrochemical approach to directly measure thedynamic process of H2O2 release from cultures of Arabidopsis thaliana cells is reported. This approach is based on H2O2 oxidation on a Pt electrode in conjunction with continuous measurement of sample pH. For [H2O2] <1mM, calibration plots were linear and the amperometric response of the electrode was maximum at pH 6. At higher concentrations ([H2O2] >1mM), the amperometric response can be described by Michaelian-type kinetics and a mathematical expression relating current intensity and pH was obtained to quantitatively determine H2O2 concentration. At pH 5.5, the detection limit of the sensor was 3.1 μ M (S/N = 3), with a response sensitivity of 0.16 AM-1cm-2 and reproducibility was within 6.1% in the range 1-5 x 10-3M (n = 5). Cell suspensions under normal physiological conditions had a pH between 5.5-5.7 and H2O2 concentrations in the range 7.0-20.5 μM (n=5). The addition of exogenous H2O2, as well as other potential stress stimuli, was made to the cells and the change in H2O2 concentration was monitored. This real-time quantitative H2O2 analysis is a potential marker for the evaluation of oxidative stress in plant cell cultures.